# BME100 f2014:Group1 L5

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# OUR TEAM

 Name: Hawley Helmbrecht Name: Sarah FakhouryRole(s) Name: Prerna GuptaRole(s) Name: Jonathan Riecker Name: Michael PinedaRole(s) Name: Timothy Black

# LAB 5 WRITE-UP

## Procedure

Smart Phone Camera Settings

• Type of Smartphone: Samsung S3
• Flash: No Flash
• ISO setting: 800
• White Balance: Auto
• Exposure: + 2.0
• Saturation: No setting to affect Saturation
• Contrast: Auto Contrast Off

Setup for Smart Phone Fluorimeter

Calibration

Place the phone in the cradle with the camera lens lined up so that it shows a view of the slide from the side (edge-on). Once the camera is set up in the cradle and lined up with the side of the slide, adjust the slide so that the light illuminates the center of the drop and the drop focuses light on the other side of it. After adjusting the slide itself, change the distance of the smartphone from the fluorimeter in order to keep the drop in focus but keep the view close.

• Distance between the smart phone cradle and drop = 5 cm

Solutions Used for Calibration

 Concentration of 2X Calf Thymus DNA solution(micrograms/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR GREEN I Dye Solution (μL) Final DNA concentration in SYBR Green I solution (μg/mL) 5 80 80 2.5 2 80 80 1 1 80 80 0.5 0.5 80 80 0.25 0.25 80 80 0.125 0 80 80 0

Placing Samples onto the Fluorimeter

1. Find the rough side of the slide (making sure to only touch the slide while wearing gloves)
2. Place the slide, with the rough side up, in the fluorimeter
3. Locate the two clear circles in the middle of the slide and add 80 μL of SYBR Green I solution
4. On the same circle with the SYBR Green I solution, place 80 μL of Sample/Calibration drops
5. Adjust the slide so that the middle of the drop is illuminated by the light and the drop becomes focused on the side

## Data Analysis

Representative Images of Negative and Positive Samples

Positive Control:

Negative Control:

Image J Values for All Calibrator Samples

Solutions Used for Products

 PCR Product Label Tube Volume of the DILUTED PCR Product Solution (μL) Volume of the SYBR GREEN I Dye Solution (μL) Dilution 1 Dilution 2 Total Dilution (Simplified Fraction) Positive 80 80 1/6 1/2 1/12 Negative 80 80 1/6 1/2 1/12 G1 1-1 80 80 1/6 1/2 1/12 G1 1-2 80 80 1/6 1/2 1/12 G1 1-3 80 80 1/6 1/2 1/12 G1 2-1 80 80 1/6 1/2 1/12 G1 2-2 80 80 1/6 1/2 1/12 G1 2-3 80 80 1/6 1/2 1/12

Calibration curve

PCR Results Summary

 PCR Product Label Tube Mean Rawintden Drop-Background PCR Product Concentration(μg/mL) Initial PCR Product Concentration(ug/mL) Positive 5305678 1.19 .0992 Negative 1928602 .23 .0192 G1 1-1 5306397 1.19 .0992 G1 1-2 6833849 1.63 .1358 G1 1-3 5792721 1.33 .1108 G1 2-1 1287212 .048 .0040 G1 2-2 3698137 .735 .0613 G1 2-3 1883281 .218 .0182
• Our positive control PCR result was 1.19 μg/mL
• Our negative control PCR result was .23 μg/mL

Observed results

• Patient 43236 : The concentration of this sample was 1.38ug/mL. The drop of sample when put into the SYBR I turned a bright green and was easily visible by the naked eye.
• Patient 55773 : The concentration of this sample was .33ug/mL. The drop of sample when put into the SYBR I stayed fairly clear and did not glow as green.

Conclusions

• Patient 43236 :This patient was very close to the positive control. This would mean that this patient does have the disease because the genetic sequence in question amplified to the same extent as the positive control.
• Patient 55773 :This patient was very close to the negative control. This leads us to believe that the patient does not have the disease because the genetic sequence in question did not amplify as much and instead only created as much PCR product as the negative control.